Gastric submucosal tissue as a novel diagnostic tool

ABSTRACT

A cell culture growth substrate comprising submucosal tissue of a warm-blooded vertebrate and a method for culturing fastidious organisms is described. Submucosal tissue used in accordance with the present invention supports the proliferation of cells when said cells are contacted with submucosal tissue under conditions conducive to cell proliferation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/775,647, filed Feb. 10, 2004, which is continuation of U.S.application Ser. No. 09/990,906, filed Nov. 14, 2001, now U.S. Pat. No.6,696,270, which is a continuation of U.S. application Ser. No.09/319,841 filed Jun. 10, 1999, which is a U.S. national application ofinternational application Serial No. PCT/US97/22729 filed Dec. 10, 1997,which claims priority to U.S. provisional application Ser. No.60/032,686 filed Dec. 10, 1996.

FIELD OF THE INVENTION

The present invention relates to a submucosal tissue composition and theuse of those compositions to promote growth of fastidious cells. Moreparticularly, the present invention is directed to the use of submucosaltissue cell culture substrates to enhance the in vitro growth, and thusthe identification and diagnosis, of vertebrate infections agents.

BACKGROUND OF THE INVENTION

Bacteria are a diverse group of organisms that live in a broad varietyof environments. In particular, many bacterial species live both on andin vertebrate hosts. Bacteria that colonize the interior spaces ofvertebrates typically exhibit specific interactions with the tissuesthat comprise the bacteria's optimal habitat in the host organism. Manybacteria express adhesions having fine tuned specificities forinteracting with eukaryotic cell-surface proteins or carbohydratestructures so that only a restricted range of hosts and tissue thatcarry the appropriate receptors are available for bacterialcolonization.

For example if bacteria cannot adhere to the mucosal layer of thevertebrate digestive system they will be removed rapidly by the localnon-specific host-defense mechanisms (peristalsis, ciliary action andturnover of the epithelial cell populations and mucus layer). Inaddition, competition between bacteria for space and nutrients, andbacteria tolerance of biochemical parameters such as pH andantimicrobial peptides, select for bacterial species/strain's that cancolonize specific niches. The result of this selective process is oftenreferred to as tissue tropism.

New strains of bacteria are continually being discovered as techniquesfor their detection improve. However, many detected strains have provento be difficult to culture outside their natural micronenvironment dueto the unique culture conditions required by these organisms.Accordingly, researchers attempting to culture microorganisms try toprovide an in vitro microenvironment which mimics the in vivoenvironment in which the microorganisms grow. The ability to propagatemicroorganisms in vitro is of particular importance for theidentification of infectious and pathogenic organisms, and for diagnosisof diseases. In addition, an in vitro microenvironment which mimics thein vivo environment enables the study of such organisms in vitro.

Many infectious agents when placed on existing culture media often failto grow and therefore are not detected, given the state of the presenttechnology. One medically significant organism that has proven difficultto culture in vitro is Helicobacter pylori, a gram negative spiralshaped microaerophilic bacteria. H. pylori live in the mucous layerlining the stomach of vertebrate species and are partially protectedfrom the stomach's acid by the mucosal layer. The organisms secreteproteins that interact with the stomach's epithelial cells and attractphagocytic cells, such as macrophages and leukocytes, and thosephagocytic cells induce inflammation and gastritis. In addition, thebacteria produce urease, an enzyme that helps to break down urea intoammonia and carbon dioxide. Ammonia can neutralize stomach acid allowingfurther proliferation of H. pylori. H. pylori also secretes toxins thatcontribute to the formation of stomach ulcers. H. pylori has beensuggested to be a causative agent of chronic active gastritis andgastric duodenal ulcers. More recently, H. pylori infections have alsobeen associated with the development of gastric adenocarcinoma andmucosa-associated lymphoid tissue lymphoma of the stomach.

Many bacteria cannot survive in an acidic environment, however H. pyloriare not the only bacteria capable of colonizing the surface of aprimate's stomach. Since the discovery of H. pylori bacteria, scientistshave isolated 11 other organisms from the stomachs of other primatessuch as dog, cats, rodents, ferrets and even cheetahs. These bacteria,for now are considered to be members of the Helocobacter family. All arespiral shaped and highly mobile, properties that enable them to resistmuscle contractions that regularly empty the stomach. These organismsgrow best at oxygen levels of 5%, matching the level found in thestomachs mucus layer (ambient air is 21% oxygen).

Surveys using an antibody-based blood test to reveal the presence H.pylori have indicated that one-third to one-half of the world'spopulation carry H. pylori. In the United States and Western Europechildren rarely become infected, but the bacteria's prevalence riseswith age such that more than half of all sixty year olds in thosecountries have the bacteria. In contrast, sixty to seventy percent ofthe children in developing countries show positive test results by age10, and the infection rate remains higher for adults. H. pyloriinfection is also common in institutionalized children. H. pylori iscapable of long term persistence in untreated individuals, and in theabsence of treatment H. pylori remains persistent in the gastric mucosafor the lifetime of the host.

Although blood tests are useful for an initial screen for detecting theexistence of an H. pylori infection, The blood test is based ondetecting antibodies to H. pylori and thus is not a direct test for thepresence of viable H. pylori bacteria. Screening for antibodies onlyprovides information on whether the individual has been exposed to H.pylori. Furthermore, the blood test is know to give false positives. Thepresent invention describes a direct assay for the presence of H. pylorithat utilizes a unique cell culture matrix to grow H. pylori.

In 1983 H. pylori were cultured in vitro for the first time by using acomplex media (Walkers media) and extending the culture timeperiods (5days instead of the normal 2 day culture). This remains the currentmethod for growing H. pylori, and accordingly, the method suffers thedisadvantage of requiring long incubation times and the use of expensivecomplex media formulations. Furthermore, the presently used culturemedia fail to mimic the natural in vivo environment of H. pylori.Cellular morphology and metabolic activity of cultured cells areaffected by the composition of the substrate on which they are grown.Presumably cultured cells function best (i.e. proliferate and performtheir natural in vivo functions) when cultured on substrates thatclosely mimic their natural environment. Therefore, current studies ofcellular function that are based on the in vitro growth of H. pylori,are limited by the lack of cell growth substrates that present theappropriate physiological environment for proliferation and developmentof the cultured cells. The compositions of the present invention providea more appropriate physiological growth environment than is currentlyavailable for growing cells that naturally occupy the stomachs ofvertebrate species.

SUMMARY OF THE INVENTION

The present invention is directed to an extracellular matrix, comprisingvertebrate submucosal tissues, that provides the necessarymicroenvironment to allow the in vitro culture of fastidious organisms.Naturally occurring extracellular matrices have the ability to serve asa substrate for the growth of prokaryotic organisms, fungal agents andless well defined infectious agents of different genus and species whichare difficult to grow in standard culture media. The extracellularmatrixes of the present invention enhance the growth, and thus thedetection of, and diagnosis of disease states caused by, theseorganisms. Likewise, cell culture substrates comprising submucosaltissue isolated from the stomach, the urinary tract, or the intestinescan be utilized to mimic the natural in vivo environments of thosetissue source organs. Thus cells growing on such substrates in vitrowill provide more physiologically relevant data regarding thesusceptibility of these organisms to various potential therapeuticagents.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “contacting” as used herein with reference to cell culture isintended to include both direct and indirect contact, for example influid communication, between the submucosal tissue and the culturedcells.

The term “conditions conducive to fastidious cell growth” and“conditions conducive to prokaryote cell growth” as used herein refer tothe environmental conditions, such as sterile technique, temperature andnutrient supply, that are considered optimal for the growth of thosecells. Typically those conditions will mimic the conditions the cellsare exposed to in their natural habitats.

The term “fastidious organism” or “fastidious cell” as used hereinrefers to organisms or cells that fail to grow (or grow very slowly) onstandard growth substrates. In particular, fastidious organisms includeprokaryotes, fungal agents and less well defined infectious agents ofdifferent genus and species which are difficult to grow in standardculture media.

There is provided in accordance with this invention a method andcomposition for supporting the proliferation, in vitro, of fastidiousorganisms. Generally the method comprises the step of contacting thefastidious cells, in vitro, with a vertebrate submucosa-derived matrixunder conditions conducive to the growth of those cells.

The submucosa derived matrices for use in accordance with the presentinvention comprise highly conserved collagens, glycoproteins,proteoglycans, and glycosaminoglycans in their natural configuration andnatural concentration. The submucosal tissue for use in this inventioncan be obtained from various organ sources, including stomach, bladderor intestinal tissue harvested from animals raised for meat production,including, for example, pigs, cattle and sheep or other warm-bloodedvertebrates. This tissue is normally a discarded by-product of meatprocessing. The tissue can be used in either its natural configurationor in a comminuted or partially digested fluidized form. Vertebratesubmucosal tissue is a plentiful by-product of commercial meatproduction operations and is thus a low cost cell growth substrate,especially when the submucosal tissue is used in its native sheetconfiguration.

In general submucosal tissue is prepared from warm-blooded tissuesincluding the alimentary, respiratory, intestinal, urinary or genitaltracts by delaminating the submucosa from both the smooth muscle layersand the mucosal layers. The preparation of intestinal submucosa isdescribed and claimed in U.S. Pat. No. 4,902,508, the disclosure ofwhich is expressly incorporated herein by reference. Urinary bladdersubmucosa and its preparation is described in U.S. Pat. No. 5,554,389,the disclosure of which is expressly incorporated herein by reference.Stomach submucosa has also been obtained and characterized using similartissue processing techniques.

UBS graft material is typically prepared from bladder tissue harvestedfrom animals raised for meat production, including, for example, pigs,cattle and sheep or other warm-blooded vertebrates. Thus, there is aninexpensive commercial source of urinary bladder tissue for use inpreparation of the tissue compositions in accordance with the presentinvention.

The preparation of UBS from a segment of urinary bladder is similar tothe procedure for preparing intestinal submucosa detailed in U.S. Pat.No. 4,902,508, the disclosure of which is expressly incorporated hereinby reference. A segment of urinary bladder tissue is first subjected toabrasion using a longitudinal wiping motion to remove both the outerlayers (particularly the abluminal smooth muscle layers) and the luminalportions of the tunica mucosa layers—the epithelial layers). Theresulting submucosa tissue has a thickness of about 80 micrometers, andconsists primarily (greater than 98%) of a cellular, eosinophilicstaining (H&E stain) extracellular matrix material. Occasional bloodvessels and spindle cells consistent with fibrocytes are scatteredrandomly throughout the tissue. Typically the UBS is rinsed with salineand optionally stored in a frozen hydrated state until used as describedbelow.

In accordance with one embodiment, the cell culture substrates comprisestomach submucosa derived from stomach tissue of a warm-bloodedvertebrate. The wall of the stomach is composed of the following layers:the tunica mucosa (including an epithelium layer, a tunica propria layerconsisting of reticular or fine areolar tissue, and a glandular layer),the tunica submucosa layer (composed of areolar tissue and lackingglands), the tunica muscularis layer (composed of three layers ofmuscle), and the serosa (a layer of mesothelium outside the looseconnective tissue which invests the muscle layers). Blood vessels,lymphatic tissue and neurological tissue also pervade the stomachtissues including the tunica submucosa.

Stomach submucosal tissue in accordance with the present inventioncomprises stomach submucosa delaminated from the glandular portion ofthe tunica mucosa and the smooth muscle layers of the muscularisexterna. The composition has proven to have the ability to induce cellgrowth and proliferation in vitro, when used as a growth substratematerial. In particular a cell substrate comprising stomach submucosaltissue has been found to enhance the in vitro growth of organisms thatnaturally inhabit the stomach of primates. Furthermore the material canserve as a useful tool for evaluating the natural patterns of growth andproliferation of culture of pathogenic organisms thus allowing bettercharacterization of the pathogenesis of the disease process.

The submucosal cell culture substrates in accordance with one embodimentof the present invention comprises stomach submucosa of a warm-bloodedvertebrate delaminated from adjacent stomach tissue layers. In oneembodiment the stomach submucosa is prepared from the stomach tissue ofprimates or other acid producing tissues of vertebrate digestive tracts.

In accordance with one embodiment, the present submucosal cell culturesubstrates comprise submucosa delaminated from the smooth muscle layersof the muscularis externa and at least the luminal portion of themucosal layer of a segment of the stomach of a warm-blooded vertebrate.In one embodiment, the submucosal tissue compositions comprise thetunica submucosa and basilar portions of the tunica mucosa of thestomach of a warm blooded vertebrate. Typically the delaminationtechnique described below provides a tissue composition consistingessentially of stomach submucosa. Those compositions are referred toherein generically as stomach submucosal tissue.

The preparation of stomach submucosal tissue from a segment of stomachis similar to the procedure for preparing intestinal submucosal tissueas detailed in U.S. Pat. No. 4,902,508, the disclosure of which isexpressly incorporated herein by reference. A segment of stomach tissueis first subjected to abrasion using a longitudinal wiping motion toremove the outer layers (particularly the smooth muscle layers) and theluminal portions of the tunica mucosa layers. The resulting submucosatissue has a thickness of about 100 to about 200 micrometers, andconsists primarily (greater than 98%) of acellular, eosinophilicstaining (H&E stain) extracellular matrix material. Occasional bloodvessels and spindle cells consistent with fibrocytes are scatteredrandomly throughout the tissue. Typically the submucosa is rinsed withwater for approximately 2 hours and optionally stored in a frozenhydrated state until used as described below.

Fluidized submucosal tissue can be prepared in a manner similar to thepreparation of fluidized intestinal submucosa, as described in U.S. Pat.No. 5,275,826 the disclosure of which is expressly incorporated hereinby reference. The submucosal tissue is comminuted by tearing, cutting,grinding, shearing and the like. Grinding the submucosal tissue in afrozen or freeze-dried state is preferred although good results can beobtained as well by subjecting a suspension of submucosal tissue piecesto treatment in a high speed (high shear) blender and dewatering bycentrifuging and decanting excess water. Additionally, the comminutedfluidized tissue can be solubilized by enzymatic digestion of thesubmucosal tissue including the use of proteases, such as trypsin orpepsin, or other appropriate enzymes or mixtures of enzymes, for aperiod of time sufficient to solubilize said tissue and form asubstantially uniform or homogeneous solution.

The present invention also contemplates the use of powder forms ofsubmucosal tissue. In one embodiment a powder form of submucosal tissueis prepared by pulverizing submucosal tissue under liquid nitrogen toproduce particles ranging in size from 0.1 to 1 mm². The particulatecomposition is then lyophilized overnight and sterilized to form a solidsubstantially anhydrous particulate composite. Alternatively, a powderform of submucosal tissue can be formed from fluidized submucosa bydrying the suspensions or solutions of comminuted and/or partiallydigested stomach submucosa.

The present submucosal tissue compositions may be sterilized usingconventional sterilization techniques including tanning withglutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the graft is preferably used. For instance, itis believed that strong gamma radiation may cause loss of strength inthe graft material. Because one of the most attractive features of thesubmucosa grafts is their ability to induce host-remodeling responses,it is desirable not to use a sterilization approach which will detractfrom that property. Preferred sterilization techniques include exposingthe graft to peracetic acid, low dose gamma irradiation (≦2.5 mRad) andgas plasma sterilization; peracetic acid sterilization being the mostpreferred method. Typically, after the tissue graft composition has beensterilized, the composition is wrapped in a non-porous plastic wrap andsterilized again using ethylene oxide or gamma irradiation sterilizationtechniques.

The submucosal tissue compositions of the present invention are used inaccordance with this invention in a method and composition for promotingthe growth and proliferation of fastidious cell cultured in vitro.Generally the method comprises the step of contacting fastidious cells,in vitro, with a vertebrate submucosa-derived matrix under conditionsconducive to cell growth. Although optimum cell culture conditions usedfor culturing cells, such as prokaryotes, depend somewhat on theparticular cell type, cell growth conditions are generally well known inthe art.

Stomach submucosal tissue of warm blooded vertebrates is one preferredsource of the cell culture substrate for use in this invention.Applicants have discovered that compositions comprising stomachsubmucosal tissue can be used for supporting growth or proliferation offastidious prokaryotic cells in vitro. Stomach submucosal tissue can beused in accordance with this invention as a cell growth substrate in avariety of forms, including its native sheet-like configuration, as agel matrix, as a supplemental component in art-recognized cell/tissueculture media, or as coating for cultureware to provide a morephysiologically relevant substrate that supports and enhances theproliferation of cells in contact with the submucosal matrix. Thesubmucosal tissue is preferably sterilized prior to use in cell cultureapplications.

In one preferred embodiment fastidious prokaryotic cells, such as H.pylori, are seeded directly onto sheets of vertebrate stomach submucosaltissue under conditions conducive to prokaryotic cell proliferation. Theporous nature of stomach submucosal tissue allows diffusion of cellnutrients throughout the submucosal matrix. Thus, for example, cells canbe cultured on either the luminal or abluminal surface of the stomachsubmucosal tissue. The luminal surface is the submucosal surface facingthe lumen of the organ source and typically adjacent to an inner mucosalayer in vivo whereas the abluminal surface is the submucosal surfacefacing away from the lumen of the organ and typically in contact withsmooth muscle tissue in vivo.

To analyzing in vitro the effect of varying cell growth conditions onthe growth characteristics of the cultured cells, the cells are seededon a cell growth substrate comprising stomach submucosal tissue of awarm-blooded vertebrate and provided a culture medium containingnutrients necessary to the proliferation of said cells. The seeded cellsare then cultured under a preselected variable cell growth condition forvarying lengths of time and then the mucosal tissue substrate and thecell population on the substrate are histologically examined. Theselected growth condition can be the presence or concentration of a cellgrowth modifier compound, such as cytokines or cytotoxic agents, in thenutrient medium. Alternatively, the selected growth condition may be themodification of environmental factors such as temperature, pH,electromagnetic radiation, or nutrient composition. The effect of theselected growth condition on the morphology and growth of the cells canthen be assessed by histological analysis of the control (cells culturedin the absence of the selected growth condition) and the test cellcultures.

In another embodiment of the present invention, cell growth substratesin accordance with the present invention are formed from fluidized formsof submucosal tissue that have been solubilized by enzymatic digestion.The fluidized, digested submucosal tissue can be gelled to form a solidor semi-solid matrix, for example, stomach submucosal tissue can befluidized, enzymatically digested and gelled to form a gelled cellculture substrate comprising stomach submucosal tissue. The viscosity offluidized submucosa for use in accordance with this invention can bemanipulated by controlling the concentration of the submucosa componentand the degree of hydration. The viscosity can be adjusted to a range ofabout 2 to about 300,000 cps at 25° C. Higher viscosity formulations,for example, gels, can be prepared from the submucosa digest solutionsby adjusting the pH of such solutions to about 6.0 to about 7.4.Eukaryotic or prokaryotic cells can then be seeded directly on thesurface of the matrix and cultured under conditions conducive to cellproliferation.

The cell growth substrate of the present invention can be combined withnutrients, including minerals, amino acids, sugars, peptides, proteins,or glycoproteins that facilitate cellular proliferation. In onepreferred embodiment fluidized or powder forms of submucosal tissue canbe used to supplement standard culture media to enhance the standardmedia's capacity for sustaining and inducing the proliferation offastidious cells cultured in vitro.

In accordance with the present invention there is provided a cellculture composition for supporting growth, in vitro, of fastidiousorganisms (including both eukaryotic and prokaryotic organisms), thecomposition comprising submucosal tissue of a warm-blooded vertebrate.The composition may further comprise added nutrients, and/or growthfactors required for optimal growth of the cultured cells. The submucosasubstrates of the present invention can be used with commerciallyavailable cell culture liquid media (both serum based and serum free).When grown in accordance with this invention, proliferating cells caneither be in direct contact with the submucosal tissue or they cansimply be in fluid communication with the submucosal tissue.

EXAMPLE 1 Preparation of Stomach Submucosal Tissue

The tissue graft material of this invention is prepared in accordancewith the following steps:

The stomach is first removed from the animal source by cutting theesophagus and small intestine at their respective entrance and exitpoints on the stomach. Any excess mesentery tissue or fat is removedfrom the stomach and the contents of the stomach are emptied and anyremaining residues are removed from the inside of the stomach by rinsingwith running tap water.

The stomach is then everted to expose the inside layers of the stomach.The portions of the stomach that begin to form the entrance or exitpoints of the stomach are removed. The stomach is typically left whole,however the stomach can also be cut and flattened prior to removal ofunwanted tissues. The luminal surface of the stomach is subject toabrasion using the handle portion of a pair of scissors or hemostats toscrape off the inner layers of the stomach including at least theluminal portion of the tunica mucosa. A thin residual layer will remainat this point. If the tissue was left whole, the stomach tissue iseverted again to return the luminal surface of the stomach to theinterior of the graft construct. A small cut is then made in theexterior muscle fiber layer. The muscle layers are then delaminated fromthe submucosal tissue through the use of a pair of scissors or hemostatto enlarge the cut in the muscle and scrape off the muscle layers.

The remaining tissue is everted again to place the luminal side on theexterior of the tissue graft. The luminal surface is scraped to removethe remaining inside residue which has a brownish color. The stomachtissue is scraped until the tissue appears pinkish-white in color.During the preparation of the stomach tissue care is taken to keep thetissue by periodically hydrating the tissue with water. The stomachsubmucosa tissue is rinsed in running tap water for approximately twohours to remove any blood or loose tissue scrapings. After rinsing thetissue should appear white, if the tissue remains pinkish in color thetissue is rubbed under water until the tissue appears white. Afterrinsing is complete excess water is removed by ringing the tissue byhand or the use of mechanical ringers. The tissue is then stored inliquid nitrogen at −80° C.

EXAMPLE 2 In-Vitro Cell Growth Properties of Stomach Submucosa

The ability of stomach submucosa to serve as an extracellular matrix tosupport in-vitro cell growth was tested by applying several cell typesto the stomach submucosal tissue surface under standard cell cultureconditions. The cell types tested included 3T3 fibroblasts, intestinalepithelium cells, and FR (fetal rat) mesenchymal cells. All three celltypes showed the ability to proliferate readily upon this extracellularmatrix without the addition of the supplements that would be needed togrow these cells on a plastic surface. Therefore, it can be concludedthat the material contains necessary structure and composition“nutrients” to serve as a cell culture substrate for supporting cellgrowth.

EXAMPLE 3 Kirby-Bauer Test

To determine if stomach submucosa inhibits the growth of H. pylori, aKirby-Bauer test was conducted. Individual colonies of H. pylori wereisolated from a chocolate agar plate and used to inoculate a 1 ml.solution of sterile saline in a small tube. This sterile saline solutionwas then used to inoculate a chocolate agar plate through the use of asterile cotton swab. A small piece of stomach submucosal tissue(approximately 25-50 mm. in diameter) was placed in the middle of theinoculated chocolate agar plate and pressed onto the surface of theplate to assure that the submucosa tissue sticks to the chocolate agar.The experiment was conducted in duplicate; two plates having the luminalside of the submucosa tissue in contact with the chocolate agar, and twoplates having the abluminal surface of the submucosa tissue in contactwith the agar. The plates were then incubated in a Campy jar in the 37°C. aerobic incubator for 3-4 days. After incubation the plates wereremoved from the incubator and Campy jar. The plates were observed todetermine if there was a zone of inhibition surrounding the submucosalmembranes. The Kirby-Bauer results show that stomach submucosal does notinhibit the growth of H. pylori or other organisms.

EXAMPLE 4 In-Vitro Growth of H. Pylori on Stomach Submucosa Substrates

In a sterile hood, 2 mL of sterile saline was transferred to a smalltube, and the saline was inoculated with H. pylori by swabbing 2chocolate agar plates containing H. pylori and transferring the bacteriato the sterile saline. Chocolate agar is a commonly used rich agarmedium comprising:

Pancreatic digest of casein: 7.5 g Selected meat peptone: 7.5 g Cornstarch 1.0 g Dipotassium phosphate 4.0 g Monopotassium phosphate 1.0 gSodium chloride 5.0 g Agar 12.0 g  Hemoglobin 10.0 g  Iso Vitalexenrichment   10. ml100 μl of the inoculated 2 ml saline solution was transferred into 5 mLof sterile saline for the McFarland test to determine cellconcentration. (5×10⁷)(5.1)=0.1A and thus A=2.55×10⁹ organisms/mL.

Serial dilutions of the H. pylori-containing saline solution were thenprepared as follows: 300 μl from the inoculated 2 mL solution was addedto 2700 μl of Walker's media giving a 10⁻¹ concentration. Then, 300 μlof the 10⁻¹ concentration solution was added to 2700 μl of Walker'smedia to give a 10⁻² concentration. This method is continued until the10⁻⁵ concentration is reached.

A sheet of stomach submucosa, having the luminal side up, was placed onthe top of a lid from a 24 well tray. The lids of a separate sterile 24well tray were marked with the labels of the appropriate controls andsamples. The stomach submucosal tissue was cut into ˜1 cm pieces using ascissors sterilized with alcohol and flamed, and the individual pieceswere transferred into the appropriate marked wells using forcepssterilized with alcohol and flamed. Stomach submucosal tissue was placedinto the wells either with the luminal side facing up or with theluminal side facing down. 400 ml of the appropriate concentration(dilution) of media plus bacteria (10⁻³, 10⁻⁴, 10⁻⁵) was added to eachof the wells. In addition, a series of “submucosa only control” wellswere prepared, having only 400 μl of sterile saline added to thesubmucosal tissue containing wells. Finally, transfer media plusbacteria only (no submucosal tissue) was added to the appropriate wellsas “bacteria only” controls.

The microliter tray was placed into a Campy® pouch and incubate for 24hours at 37° C. in an aerobic (O₂) incubator. After incubation sampleswere taken from both the supernatant and the membrane of each of themicrotiter wells and plated onto chocolate agar plates. Two samples wereplated per concentration for dilutions up to 10⁻⁵ (including theoriginal inoculated saline solution) to determine the growth efficiencyof the H. pylori cultured in the presence of submucosal tissue.

To quantitate the number of bacteria present in the submucosal tissuesupernatant, 100 μl of the supernatant was removed from the well, platedonto a chocolate agar plate and spread with an alcohol flamed hockeystick. To measure the number of H. pylori growing in and on thesubmucosal membrane, the cells were isolated from the tissue as follows:The submucosal tissue was removed from the well and cut into 2 or 3pieces. These pieces were placed into a centrifuge tube containing 400μl of fresh Walker's media and the tube was vortexed for ˜30 seconds.100 μl of the vortexed solution was plated onto a chocolate agar plateand spread with an alcohol flamed hockey stick.

The inoculated chocolate agar plates were then incubated in a Campy® jarin a 37° C. aerobic incubator for 3-4 days. The plates were removed fromthe incubator and Campy® jar and number of colonies on each plate wasdetermined by observation through a dissecting microscope. The number oforganisms per mL was calculated: Number of colonies×the dilution factor(either 10³, 10⁴, or 10⁵)×10 (for the 100 μl placed on the plate)=Thenumber of organisms per mL. For example 57 colonies on 10⁻³ dilutionplate equals 57×10³×10=5.7×10⁵ organisms/mL. The accumulated data fromthe in vitro culture of H. pylori in the presence of stomach submucosaare indicated in Table 1.

TABLE 1 Concentration Sidedness “In membrane” “In supernatant” 10⁻³Inner up A To contaminated to Fungus, Plate A count 3.16 × 10⁶  Plate BNo Growth Fungus, 2.49 × 10⁶  Inner up B 2.0 × 10⁵ Contaminated, Plate A1.38 × 10⁶  Plate B Fungus, Contaminated, 1.3 × 10⁵ TCTC Outer up AContaminated, Fungus, Contaminated, Plate A 2.4 × 10⁵ No Growth Plate BContaminated, Contaminated, 2.1 × 10⁵ TCTC Outer up B Fungus, 3.21 ×10⁶  Plate A 1.7 × 10⁵ Plate B Fungus, Fungus 2.2 × 10⁵ 3.22 × 10⁶  10⁻⁴Inner up A 4.0 × 10⁵ Contaminated, Plate A 6.3 × 10⁶ Plate B 2.0 × 10⁵Contaminated, 2.5 × 10⁶ Inner up B 3.0 × 10⁵ Contaminated, Plate A 1.7 ×10⁶ Plate B 2.0 × 10⁵ 2.5 × 10⁶ Outer up A Contaminated, Contaminated,Plate A NG NG Plate B Contaminated, Contaminated, 2.0 × 10⁵ NG Outer upB Contaminated, Fungus, 4.2 × 10⁶ Plate A 2.0 × 10⁵ Plate BContaminated, 4.3 × 10⁶ 2.7 × 10⁶ 10⁻⁵ Inner up A No Growth 7.0 × 10⁶Plate A Plate B No Growth 2.0 × 10⁶ Inner up B Fungus, 3.0 × 10⁶ Plate ANo Growth Plate B Fungus, 6.0 × 10⁶ No Growth Outer up A Fungus,Contaminated, Plate A No Growth No Growth Plate B Fungus, 2.0 × 10⁶ NoGrowth Outer up B No Growth Contaminated, Plate A 4.0 × 10⁶ Plate B NoGrowth 2.0 × 10⁶

Results

The calculated values were compared to the numbers obtained from theoriginal serial dilutions and the McFarland standard to determine theextent of the proliferation that occurred during the incubation of H.pylori in the presence of the stomach submucosal tissue. As the data ofTable 1 indicates, stomach submucosa tissue is capable of supporting H.pylori growth. The number of colonies present on the chocolate agarplates increased with each serial dilution as expected which indicatesgrowth of H. pylori. Contamination continues to be a problem in theseexperiments. Accordingly, the optimal conditions for growing H. pylorion stomach submucosa have not yet been determined. However theexperiments demonstrate stomach submucosa's ability to support H. pylorigrowth. It is anticipated that once the optimal conditions have beendefined the difficulties with contamination by other organisms will beeliminated.

The present experiment analyzed the growth of H. pylori on stomachsubmucosal tissue substrates in the presence of bacterial cell culturemedia. In an alternative embodiment, the stomach submucosal tissuesubstrates can be used to culture H. pylori in the presence ofeukaryotic cell culture media. The presence of the eukaryotic cellculture media, and most preferably mammalian cell culture media, willprovide a more physiological environment and thus optimize the functionof the stomach submucosal tissue as a host for microbial residence andpathogenicity.

Once the conditions have been optimized for growing H. pylori in vitro,the antibiotic sensitivity of these organisms can be evaluated in anenvironment that more closely mimics the natural habitat of theseorganisms. Therefore the culture substrates of the present invention canbe used not only for detecting the presence of H. pylori from a sourcetissue, but can also be utilized to investigate the optimal antibioticsand antibiotic concentrations necessary to effectively treat patientsinfected with H. pylori.

What is claimed is:
 1. A composition comprising: a sterilizedextracellular matrix material comprising submucosa, wherein theextracellular matrix material is obtained from a urinary bladder of apig, wherein the extracellular matrix material is acellular.
 2. Thecomposition according to claim 1, which has been sterilized withperacetic acid.
 3. The composition according to claim 1, wherein thesubmucosa comprises collagens, glycoproteins, proteoglycans, andglycosaminoglycans.
 4. The composition according to claim 1, whichexhibits a capacity to induce remodeling in a host.
 5. The compositionaccording to claim 1, wrapped in a non-porous plastic wrap.
 6. Thecomposition according to claim 3, in a native sheet form.
 7. Thecomposition according to claim 3, in a powder form.
 8. The compositionaccording to claim 4, which has been sterilized with peracetic acid. 9.The composition according to claim 4, wherein the submucosa comprisescollagens, glycoproteins, proteoglycans, and glycosaminoglycans.
 10. Thecomposition according to claim 3, in a gel form.